Age-related changes in cellular electrophysiology and calcium handling for atrial fibrillation

Age-related changes in cellular electrophysiology and

calcium handling for atrial fibrillation

Guo-Jun Xu a, Tian-Yi Gan a, Bao-Peng Tang a, *, Zu-Heng Chen a, Tao Jiang b, Jian-Guo Song c,Xia Guo d, Jin-xin Li d

a Department of Cardiology, First Affiliated Hospital, Xinjiang Medical University, Urumqi, Chinab Department of Animal Experiment, First Affiliated Hospital, Xinjiang Medical University, Urumqi, Chinac Laboratory of Electrophysiology, First Affiliated Hospital, Xinjiang Medical University, Urumqi, China

d Department of Molecular Biology, Xinjiang Medical University, Urumqi, China

Received: February 22, 2013; Accepted: May 20, 2013

Abstract

This study was to investigate whether or not the dysfunction of atrial repolarization and abnormality of the intracellular Ca2+ handling proteinwas augmented with ageing. Four groups of dogs were studied, adult and aged dogs in sinus rhythm (SR) and atrial fibrillation (AF) induced byrapid atrial pacing. We used whole cell patch clamp recording techniques to measure L-type Ca2+ current in cardiomyocytes dispersed from theleft atria. Expressions of the Ca2+ handling protein were measured by real-time quantitative reverse transcription-polymerase chain reaction andWestern blot methods. Cardiomyocytes from old atria showed longer action potential (AP) duration to 90% repolarization, lower AP plateaupotential and peak L-type Ca2+ current densities at both age groups in SR. AF led to a higher maximum diastolic potential, an increase of ampli-tude of phase 0, decreases of AP duration to 90% repolarization, plateau potential and peak L-type Ca2+ current densities. Compared to the adultgroup, mRNA and protein expressions of the L-type calcium channel a1c were decreased, whereas expressions of calcium adenosine triphos-phatase were increased in the aged group. Compared to SR group, expressions of Ca2+ handling protein except for phospholamban were signif-icantly decreased in both age groups with AF. We conclude that these ageing-induced electrophysiological and molecular changes showed thatgeneral pathophysiological adaptations might provide a substrate conducive to AF.

Keywords: Atrial fibrillation� Ageing� L-type Ca2+ current� Cellular electrophysiology� Ca2+ handling proteins

Introduction

It is well-established that the incidence and prevalence of atrial fibril-lation (AF) increases with age [1, 2]. Although the mechanism under-lying AF has been investigated in humans and in animal models,cellular electrophysiological and molecular changes that render theatria of aged individuals more susceptible to AF than those of adultsremain poorly understood.

Previous studies have reported that L-type Ca2+ current (ICa-L),the major action potential (AP) plateau current, is reduced in agedcanine right atrial cells compared with adults [3]. Recent evidence

has shown that AP duration (APD) in right atrial cardiomyocytes isprolonged with age and the AP plateau becomes increasingly negativewith age [4, 5]. In fact, AF mainly come from left atria, rather thanright atria [1–3, 6], as we well know in clinical practice. However,there are no published data on the effects of age on left atrial ICa-L.While it is clear that abnormal intracellular Ca2+ dynamics may under-lie electrical remodelling in specialized aged atrial cells [6–9], as yetthere has been no systematic study of intracellular Ca2+ handling inaged left atria. Some scholars have suggested that ageing reducesthe activity of proteins influencing the calcium homeostasis in persis-tent human AF [10–14]. However, because of the lack of aged controlgroup, it is less obvious whether the change is because of age, thepresence of AF or other reasons. We suggest that dysfunction of atrialrepolarization and abnormality of the intracellular Ca2+ handling pro-tein (L-type calcium channel a1c (LVDCCa1c), calcium adenosinetriphosphatase (Ca2+-ATPase), ryanodine receptor type-2 (RYR2), ino-sitol triphosphate receptor type-1(IP3R1) or ancillary proteins phos-pholamban (PLN) in the microenvironment increase with ageing, and

*Correspondence to: Bao-Peng TANG,Department of Cardiology, First Affiliated Hospital,

Xinjiang Medical, University,

LiYuShan Street, No 137, Urumqi 830011, China.

Tel.: +86-991-4366170Fax: +86-991-4366170

E-mail: [email protected]

ª 2013 The Authors.

Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use,

distribution and reproduction in any medium, provided the original work is properly cited.

doi: 10.1111/jcmm.12084

J. Cell. Mol. Med. Vol 17, No 9, 2013 pp. 1109-1118

thus create a substrate for initiation and maintenance of AF. The aimof the present study was to determine whether the general patho-physiological mechanisms of normal old atria provide a substrateconducive to atrial arrhythmias, particularly AF.

Materials and methods

Animal preparation

Fourteen adult (1–3 years) and 14 aged (more than 8 years) mongrels,

weighing 18–26 kg each, were obtained from the Animal Center (Xinji-ang Medical University, Urumqi, China). The ages of the dogs were esti-

mated by a veterinarian based on standard measures for age, including

dentition, coat, eyes and musculoskeletal and conformational descrip-

tors. The dogs were kept in a temperature-controlled house under a12 h light/dark cycle and fed a standard laboratory diet and water ad

libitum. The Animal Care and Use Committee of the Xinjiang Medical

University have approved all experiments in accordance with the Decla-ration of the National Institutes of Health Guide for Care and Use of

Laboratory Animals (Publication No. 85-23, revised 1985).

Six-lead electrocardiogram (ECG) measurements were performed on

conscious dogs resting quietly to confirm sinus rhythm (SR). Echocar-diograms were performed to exclude structural heart disease. Dogs of

each age were randomly divided into four groups of seven animals, the

adult SR group and the aged SR group, the adult AF group and the

aged AF group. AF was induced by chronic rapid atrial pacing anddefined as persistence of AF for at least 5 days.

Induction of AF

Animals were anaesthetized with pentobarbital sodium (30 mg/kg i.v.)

and ventilated with isoflurane, 1.5–2%, and O2, 2 l/min. Morphine sul-

phate 0.15 mg/kg was injected into the epidural space to maintain post-operative analgesia. Using sterile techniques, a right intercostal thora-

cotomy was performed, the pericardium was opened and the heart was

suspended in a pericardial cradle. A lead was attached to the epicar-

dium of the left atrial appendage. The lead was tunnelled subcutane-ously and connected to a Pulse Generator (Department of Electronic

Engineering, Fudan University, Shanghai, China). Pulse generators were

implanted in subcutaneous pockets on the left posterior chest wall.After the incisions were closed and the dogs recovered from anaesthe-

sia, they were monitored for 2–3 days in the recovery room before

being moved to routine care. The dogs were prophylactically treated

with cefazolin, 25 mg/kg IV twice daily for 2 days after surgery. Theywere allowed to stabilize for 1 week and then were paced from the left

atrial appendage at 600 bpm to induce persistent AF. Dogs were used

for in vitro study after they had been in persistent AF for ≥5 days.

Atrial myocyte preparation

At the end of the experiments, the dogs were anaesthetized with pento-barbital sodium (30 mg/kg i.v.) and sternotomies were performed. The

hearts were quickly removed, and parts of the left atrial wall samples

were rapidly frozen in liquid nitrogen and separately stored at �80°C

for further analysis. One aliquot of each tissue sample was used toinvestigate mRNA expression of target genes, whereas the other part

was used to determine protein levels. At the same time, their hearts

were rinsed in oxygenated Ca2+-free Tyrode’s solution (mmol/l): NaCl

137; KCl 5.4; MgCl2 1.0; NaH2PO4 0.33; HEPES 10; and Glucose 10 (pH7.4, NaOH). The aortae were cannulated and the hearts were retro-

gradely perfused on a Langendorff apparatus at 37°C. A perfusion of

Ca2+-free Tyrode’s solution for 5 min was followed by Ca2+-free Ty-rode’s solution containing 0.03% collagenase-II (Worthington Biochemi-

cal, Lakewood, CO, USA) and 1% bovine serum albumin (BSA) for

35 min. The left atrium (LA) were dissected, minced and gently tritu-

rated with a pipette in a Ca2+ Tyrode’s solution containing 1% BSA at37°C for 10 min. The cells were filtered through a 200 lm nylon mesh,

and resuspended in the Tyrode’s solution in which the Ca2+ concentra-

tion was gradually increased to 1.0 mmol/l. Only cells with rod-shaped

morphology and clear cross-striation were used for experiments.

Cellular electrophysiological studies

Cells of the LA in a 1 ml bath were continuously superfused (2–3 ml/

min.) with normal Tyrode’s solution containing (mmol/l): NaCl 137, KCl

5.4, MgCl2 1.0, CaCl2 1.8, NaH2PO4 0.33, HEPES 10 and glucose 10 (pH

was adjusted with NaOH to 7.4). The solution was bubbled with 100%O2. Membrane currents and AP were recorded using whole-cell patch-

clamp techniques with an EPC 10 Double amplifier (HEKA, Lambrecht,

Pfalz, Germany) and Patchmaster software. Patch pipette resistances

ranged from 2.0 to 3.0 MΩ, when filled with an internal solution. The APwas recorded in current-clamp mode. The solution for AP recording

(mmol/l) was NaCl 137, KCl 5.4, MgCl2 1.0, CaCl2 1.8, HEPES 10 and

Glucose 20 (pH was adjusted with KOH to 7.4). The electrode internalsolution for AP recording was KCl 140, MgCl2 2.0, egtazic acid 2.0, HE-

PES 5.0, EGTA 5 and Na2 ATP 4.0 (pH was adjusted with KOH to 7.4).

Calcium currents were recorded in the voltage-clamp mode. The external

solution for ICa-L recording contained (mmol): Choline-Cl 137, CaCl2 2.0,MgCl2 1.0, HEPES 5, Glucose 10, CsCl 4.6, TEA-Cl 10, and 4-AP 5 (pH

7.30, CsOH).The internal solution for ICa-L recording contained (mmol):

CsCl 120, MgCl2 1.0, MgATP 5.0, BAPTA 10, HEPES 10 and TEA-Cl 10

(pH 7.30, CsOH). In this study, we started data acquisition 10 min. aftermembrane rupture. ICa-L magnitudes were normalized by each cellular

membrane capacitance (pF) and expressed as current density (pA/pF).

Recordings were filtered at low pass (2 Hz) and high pass (30 Hz). Activa-tion voltage dependence was assessed from depolarization-induced cur-

rents, with driving force corrected by dividing TP-Erev, where Erev is the

voltage axis intercept of the ascending limb of the current-voltage relation.

Inactivation was assessed with 1-sec. prepulses from �60, �50, �40,�35, �30, �25, �20, �15, �10, 0, 10, 20, 30 and 40 mV, followed by

240-ms test pulses to +10 mV. The Boltzmann equation was used to fit

data. ICa-L recovery was studied with paired 240-ms pulses to 10 mV

(0.1 Hz) delivered at a progressively increasing interpulse interval (P-P)ranging from 3, 5, 8, 10, 20, 40, 60, 80, 160, 300, 500 to 1000 ms.

Detection of gene expression

Total RNA was extracted from samples of the LA free wall using TRI-

ZOL(Invitrogen Life Technologies, Carlsbad, CA, USA). Expression levels

of target genes were measured by real-time quantitative reverse tran-scription-polymerase chain reaction (qRT-PCR) using SybrGreen qPCR

1110 ª 2013 The Authors.


Master Mix (Bio-Rad, Hercules, CA, USA) in a 20 ll reaction volumecontaining 50 ng of cDNA. All reactions were performed in triplicate

and included negative controls. PCR were carried out using an ABI

Prism 7500 Sequence Detection System (Applied Biosystems, Carlsbad,

CA, USA). Cycling conditions were as follows: 2 min. at 50°C, 10 min.at 95°C, and 40 cycles of 15 sec. at 95°C and 1 min. at 60°C. Relativequantification of mRNA levels was obtained by the 7500 system soft-

ware using comparative methods. Fluorescence signals were normalizedto the housekeeping gene b-actin. The comparative threshold-cycle (CT)

relative quantification method was used (DDCT). For each sample, each

gene was quantified in duplicate in three separate experiments. The val-

ues were averaged and then used to calculate 2�DDCT, which corre-sponds to the expression relative to b-actin. The expected size

amplicons were confirmed by gel electrophoreses. The sequences of

the genes studied were obtained from GenBank, and the primers were

designed using Primer 5.0 software (Applied Biosystems). The ampliconsize of the primer sequence and annealing temperature of the genes are

shown in Table 1.

Assessment of protein expression

Membrane protein was extracted from tissue samples of LA with

5 mmol/l Tris-HCl (pH 7.4), 2 mmol/l ethylenediamine tetraacetic acid(EDTA), 5 lg/ml leupeptin, 10 lg/ml benzamidine and 5 lg/ml soybean

trypsin inhibitor, followed by tissue homogenization. All procedureswere performed at 4°C. Equal amounts (100 lg/sample) of LA mem-

brane proteins were separated on 8% sodium dodecyl sulphate-poly-

acrylamide gel electrophoresis (SDS-PAGE) gels and transferred to

polyvinylidine difluoride membranes. Membranes were blocked in 5%non-fat dry milk in TTBS (Tris-HCl 50-mmol/l, NaCl 500-mmol/l; pH 7.5,

0.05% Tween-20) for 2 hrs at room temperature and then incubated

with primary antibody (1:500 dilution) in 5% nonfat dry milk in TTBSfor 4 hrs at room temperature. Membranes were then incubated with

the following antibodies: rabbit polyclonal anti-Cav1.2 (LVDCCa1c; Santa

Cruz Biotechnology Inc, Santa Cruz, CA, USA), rabbit polyclonal anti-

IP3R1 (Santa Cruz Biotechnology), rabbit polyclonal anti-Ca2+-ATPase(Santa Cruz Biotechnology), rabbit polyclonal anti-PLN (Abcam, Cam-

bridge, MA, USA) and mouse monoclonal anti-RYR2 (Abcam). Mem-

branes were washed three times in TTBS, reblocked in 5% non-fat dry

milk in TTBS (15 min.) and then incubated with horseradish peroxidase-conjugated goat anti-rabbit or goat antimouse IgG secondary antibody

(1:5000) in 5% non-fat dry milk in TTBS (40 min.). Immunoreactive

bands were detected by Immun-Star horseradish peroxidase (HRP) sub-strate (Bio-Rad) and quantified by densitometry analysis using an Image

Quant 350 imager and Image Quant TL-1 software (GE Healthcare, Fair-

field, CT, USA). Anti-b-actin antibody (Santa Cruz Biotechnology) was

used to control for equal protein loading and to normalize ion channelprotein band intensity. All Western blot target bands were quantitatively

expressed by normalization to the control band on the same lane. Wes-

tern blot band intensities were expressed as optical density (OD) units

corresponding to densitometric band intensity following backgroundsubtraction divided by b-actin signal intensity for the same sample.

Statistical analysis

Action potential characteristics measured were maximum diastolic poten-

tial (MDP), amplitude of phase 0 (APA), plateau potential and APD to 90%

repolarization (APD90). Quantitative data were presented as mean � SD.Comparisons between the quantitative data were made using ANOVA.

P < 0.05 was considered statistically significant. Software SPSS 15.0 was

used for statistical analysis (SPSS Inc., Chicago, IL, USA).

Results

ECG data

The ECGs of the old dogs manifested longer P-wave durations and P-wave dispersion than adults (66.1 � 6.4 ms versus 75.9 � 5.3 ms;19.1 � 4.1 ms versus 26.7 � 3.1 ms, n = 7, all P < 0.05, respec-tively). Other variables did not differ. There was no difference betweenthe two groups in time to onset of persistent AF, the adult dogsdeveloped persistent AF after 40 � 5 days and old dogs after52 � 7 days of atrial pacing (P > 0.05).

Changes in AP characteristics

Representative recordings and summary data for major AP parame-ters of adult and aged dogs in SR and those with AF are shown in

Table 1 Primer sequence and amplicon size of genes

GenePrimersequence

Ampliconsize (bp)

Annealingtemperature(°C)

b-actin F: 5′-AAGGACCTGTATGCCAACACA-3′

R: 5′-ATCCACACAGAATACTTGCGTT-3′

152 57

LVDCCa1c F: 5′-GACGCTATGGGCTATGAGTTAC-3′

R: 5′-AGTCCAGGTAGCCCTTTAGGT-3′

199 58

Ca2+-ATPase F: 5′-TGGATTACAATGAGGCGAAG-3′

R: 5′-AGACCCTTCAATTCGGTATCA

112 56.5

PLN F: 5′-CACAAGAGCCAAGGCTACCT-3′

R: 5′-CAGGAAAGCAGGAAGTCTCAA-3″

135 58

RYR2 F: 5′-ATTGAGAAACGATTTGCCTACA-3′

R: 5′-GGGAAATGTTCTCCTTTGCTT-3′

116 57.5

IP3R1 F: 5′-ACGCTATGGGCTCGGTAGTTA-3′

R: 5′-ACAGAATACTTGCTTCTCCTT-3′

140 57



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J. Cell. Mol. Med. Vol 17, No 9, 2013

Table 2 AP characteristics recorded from adult and old atria in SR and AF at a cycle length of 2000 ms

Group n MDP (mv) APA (mv) Plateau (mv) APD90 (ms)

SR adult 24 �78.8 � 0.8 109.8 � 1.4 �4.0 � 0.7 320.0 � 7.9

SR aged 30 �79.2 � 1.4 110.5 � 4.9 �7.5 � 1.7* 340.5 � 10.1*

AF adult 28 �71.8 � 0.9* 121.8 � 1.1* �6.4 � 1.1* 297.0 � 5.6*

AF aged 26 �72.2 � 1.2† 122.5 � 2.9† �9.8 � 1.1† 300.5 � 7.1†

*P < 0.05, compared with the adult SR group.†P < 0.05, compared with the aged SR group.Data are presented as mean � SD.AP: action potential; MDP: maximum diastolic potential; APA: action potential amplitude; APD90: action potential duration to 90% repolarization;SR: sinus rhythm; AF: atrial fibrillation; n: the number of cells of each group (SR adult: 24 cells of seven dogs, SR aged: 30 cells of seven dogs,AF adult: 28 cells of seven dogs, AF aged: 26 cells of seven dogs).

A B

C D

Fig. 1 AP recording from LA cardiomyocytes of adult SR canines (A), aged SR canines (B), adult AF canines (C), and aged AF canines (D). AP:action potential; LA: left atira; SR: sinus rhythm; AF: atrial fibrillation. (SR adults: 24 cells of seven dogs, SR aged: 30 cells of seven dogs, AFadults: 28 cells of seven dogs, AF aged: 26 cells of seven dogs).



Table 2 and Figure 1. Cardiomyocytes from aged atria were longerAPD90, AP plateau potential was significantly lower in comparison toadults, while there were no significant differences in MDP and APA.APD90 was shortened with AF in both adult and aged groups withmore shortening in the latter resulting in no difference between APD90

in both AF groups. AF led to a higher MDP, a significant increase inAPA and a lower of AP plateau potential at both ages. AF was associ-ated with a significant depolarization of the cellular membrane in bothadult and aged LA. The extent of depolarization was the same in bothAF groups leaving adult cardiomyocytes more depolarized than agedcardiomyocytes.

Changes in ICa-L characteristics

Typical recordings of ICa-L and comparative major ICa-L parameters ofadults and aged dogs in SR and those with AF are shown in Table 3and Figure 2. Aged LA cardiomyocytes demonstrated lower peak ICa-Ldensities than adult LA cells. This decrease tendency was the same inthe both adults and aged groups in AF, while the latter appeared to belower. Activation voltage dependence had no significant difference inhalf-activation voltage and slope factor of each group; also, inactiva-tion had no significant difference in half-inactivation voltage and slopefactor of each group. Otherwise, this current reduction during ageingand in AF was unaccompanied by a significant change in its recoverytime from inactivation.

Left atrial mRNA and protein expressions ofproteins influencing calcium homeostasis

As shown in Tables 4 and 5 and Figures 3 and 4, compared to theadult group, mRNA and protein expressions of LVDCCa1c were signifi-cantly decreased, mRNA and protein expressions of Ca2+-ATPasewere significantly increased in the aged group (all P < 0.05). More-

over, mRNA and protein expressions of RYR2, IP3R1 and PLN showedup-regulation tendency in the aged group, but were not significantlygreater in the two groups (all P > 0.05).

Compared to control groups, mRNA and protein expressions ofLVDCCa1c were significantly decreased; moreover, mRNA and proteinexpressions of Ca2+-ATPase, RYR2 and IP3R1 were significantlydecreased in both adult and aged groups in AF (all P < 0.05), whilemRNA and protein expressions of PLN showed a down-regulationtendency, but had no significant difference in two groups (P > 0.05).

Discussion

Ageing-associated changes of LAelectrophysiology in SR

Interestingly, our study showed that the most remarkable alterationwith ageing was a significant lowering of the AP plateau potential andAPD90 was prolonged in aged LA cardiomyocytes. Moreover, P waveduration and dispersion were significantly longer in the aged canines.The result might be a reflection of ageing-associated degree of slowerconduction of atria. Previous studies have reported that ICa-L isreduced in aged canine right atria cardiomyocytes compared to adults[3], but there are no published data on the effects of age on LA car-diomyocytes ICa-L. Our study demonstrated that there was a signifi-cant reduction in peak ICa-L in aged canine LA cardiomyocytes, whiledecreased LVDCCa1c protein levels might be the major reason of thereduced ICa-L. However, the current reduction in aged atrial cardio-myocytes was unaccompanied by a significant change in calciumchannel availability or recovery from inactivation. The currents whichdetermine the plateau level of AP in atria are IKur, Ito and ICa-L [15, 16].Therefore, a decrease in depolarizing current ICa-L or an increase in re-polarizing currents (IKur and/or Ito) may lead to the lower plateau ofAP. So the result suggested that the decrease in ICa-L may be a major

Table 3 Electrophysiological characteristics of ICa-L between adult and aged LA in SR and AF

Group n ICa-L density (pA/pF)Steady-state activation Steady-state inactivation Monoexponential recovery

time constants (ms)V0.5 (mV) k (mV) V0.5 (mV) k (mV)

SR adult 14 �14.1 � 0.8 �7.1 � 1.5 5.7 � 0.4 �23.1 � 2.1 6.2 � 0.3 51.9 � 3.3

SR aged 16 �8.1 � 0.5* �6.7 � 2.8 5.5 � 0.5 �22.9 � 3.3 6.4 � 0.5 53.1 � 3.1

AF adult 15 �9.4 � 0.7* �6.9 � 1.2 5.1 � 0.3 �22.1 � 1.9 6.2 � 0.3 51.2 � 2.3

AF aged 19 �5.9 � 0.3† �6.8 � 2.1 5.9 � 0.3 �21.9 � 2.3 6.8 � 0.6 52.1 � 5.1

*P < 0.05, compared with the adult SR group.†P < 0.05, compared with the aged SR group.Data are presented as mean � SD.V0.5 and k are average values of voltage at half maximal availability and slope factor as determined using a Boltzmann equation. ICa-L currentdensities at maximal voltage (�90 mV) are shown.LA: left atira; SR: sinus rhythm; AF: atrial fibrillation; n: the number of cells of each group (SR adult: 14 cells of seven dogs, SR aged: 16 cellsof seven dogs, AF adult: 15 cells of seven dogs, AF aged: 19 cells of seven dogs).



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mechanism for the low plateau potential in aged canine LA cardio-myocytes. The longer APD90 in old atria suggested some ageing-induced changes of delayed rectifier potassium (IK) or might be

simply a consequence of the low plateau potential in aged dogs. Pre-vious study revealed that negative plateau potentials had a lower driv-ing force for conduction of early premature beats [17, 18].Therefore,

A

C

E

D

B

Fig. 2 ICa-L tracings between adults and aged LA in SR and AF, holding voltage of �70 mV to various test voltages, adult SR canines (A), aged SR

canines (B), adult AF canines (C), and aged AF canines (D); LA: left atira; SR: sinus rhythm; AF: atrial fibrillation. (E) Average peak ICa-L density in

adult and aged cells. All data were collected at the same time after establishing whole cell configuration (adults, 17 � 0.8 min.; aged,18 � 1.1 min.). (SR adult: 14 cells of seven dogs, SR aged: 16 cells of seven dogs, AF adult: 15 cells of seven dogs, AF aged: 19 cells of seven

dogs).



our results implied that the change in AP in old atria would lead to adecreased conduction of premature beats. Slow conduction of earlypremature impulses might well further facilitate the onset of AF.

Impact of AF on electrophysiology of adult andold LA

Many experimental studies have demonstrated that AF remodels atrialelectrophysiology to facilitate its own recurrence [19, 20]. The majorelectrophysiological characteristics of electrical remodelling arereduction in the atrial refractory period and loss of APD adaptation torate [21, 22]. To date, studies have been performed in normal adultanimals. AF-induced electrophysiological remodelling in adults resultsfrom rapid atrial activation, and rapid atrial pacing produces similarAP changes. Yet, the mechanism for AP changes seen with thisremodelling may differ, as shown in our study. In our study, we foundthat there was reduced ICa-L in persistent AF cardiomyocytes versuscontrols. The change was interpreted as resulting from reducedLVDCCa1c protein levels. It appears reasonable to propose that cal-cium ion channel remodelling is the basis of the atrial electricalremodelling of AF. On the basis of the present results, it was foundthat AF led to a remarkable shortening of APD90, a higher MDP, a sig-nificant increase in APA and a significant decrease in AP plateau

potential at both ages. AF-induced decreases in APD90 might beexplained by the reduction in ICa-L, AF was associated with a signifi-cant depolarization of the cellular membrane in both adults and agedLA. Such membrane depolarization in AF may be a consequence ofdecreased basal ICa-L.

Ageing-associated changes of molecular biologyof LA in SR and in AF

In this study, it is noteworthy that LVDCCa1c expression levels weresignificantly downregulated, however, intracellular Ca2+ handlingproteins generally showed an up-regulation tendency during ageing;moreover, the expression levels of LVDCCa1c and intracellular Ca2+

handling proteins except PLN were are highly downregulated in theadult and aged groups in AF, more specifically in the latter withAF. In the intact heart, electrical and mechanical alternans are mostfrequently observed during acute myocardia in AF, a condition thatis likely to affect glycolytic metabolism through restricted substrateavailability [23–27]. As a result, the phosphorylation reactions areslowed and the availability of active RyR2 channels on a beat-to-beat basis is reduced [28, 29]. Therefore, the hypothesis of attenu-ated energy production based on glycolytically derived ATP remainsan intriguing possibility to explain the increased susceptibility of

Table 4 The expressions of mRNA in the left atrial myocardium between adult and aged LA in SR and AF

Group n LVDCCa1c Ca2+-ATPase RYR2 IP3R1 PLN

SR adult 7 2.38 � 1.03 1.14 � 0.83 2.49 � 1.02 2.68 � 0.97 1.72 � 0.71

SR aged 7 1.17 � 0.75* 2.32 � 0.75* 3.63 � 0.89 3.12 � 1.21 1.97 � 0.84

AF adult 7 0.27 � 0.25* 0.30 � 0.12* 0.52 � 0.21* 0.85 � 0.21* 1.28 � 0.94

AF aged 7 0.10 � 0.07† 0.17 � 0.07† 0.26 � 0.09† 0.67 � 0.19† 1.46 � 0.52

*P < 0.05, compared with the adult SR group.†P < 0.05, compared with the aged SR group.Data are presented as mean � SD.LVDCCa1c: L-type calcium channel a1c; Ca2+-ATPase: calcium adenosine triphosphatase; RYR2: ryanodine receptor type-2; IP3R1: inositol tri-phosphate receptor type-1; PLN: phospholamban; LA: left atira; SR: sinus rhythm; AF: atrial fibrillation.

Table 5 The expressions of protein in the left atrial myocardium between adult and aged LA in SR and AF

Group n LVDCCa1c Ca2+-ATPase RYR2 IP3R1 PLN

SR adult 7 0.28 � 0.11 0.36 � 0.08 0.23 � 0.04 0.28 � 0.07 0.32 � 0.09

SR aged 7 0.13 � 0.10* 0.48 � 0.13* 0.26 � 0.05 0.35 � 0.06 0.36 � 0.08

AF adult 7 0.13 � 0.01* 0.25 � 0.07* 0.17 � 0.04* 0.17 � 0.01* 0.31 � 0.04

AF aged 7 0.07 � 0.05† 0.13 � 0.03† 0.10 � 0.02† 0.15 � 0.04† 0.31 � 0.05

*P < 0.05, compared with the adult SR group.†P < 0.05, compared with the aged SR group.Data are presented as mean � SD.LVDCCa1c: L-type calcium channel a1c; Ca2+-ATPase: calcium adenosine triphosphatase; RYR2: ryanodine receptor type-2; IP3R1: inositol tri-phosphate receptor type-1; PLN: phospholamban; LA: left atira; SR: sinus rhythm; AF: atrial fibrillation.



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aged atria to AF. It is known that sarcoplasmic reticulum Ca2+ cir-culation and the activity of Ca2+ handling proteins are regulated byphosphorylation processes [26, 28–30]. On the basis of our

results, it appears reasonable to propose that due to ageing,especially in the occurrence of AF, the rate of phosphorylation ofCa2+ handling proteins is slowed and the equilibrium between

Fig. 3 Representative gels of Ca2+ han-

dling protein (LVDCCa1c, L-type calciumchannel a1c; Ca2+-ATPase, calcium adeno-

sine triphosphatase; RYR2, ryanodine

receptor type-2; IP3R1, inositol triphos-

phate receptor type-1; PLN, phospholam-ban) and b-actin in the LA myocardium

between adult and aged groups in SR and

AF. LA: left atira; SR: sinus rhythm; AF:

atrial fibrillation; M: marker; AS: Adult SRgroup; OS: Old SR group; AF: Adult AF

group; OF: Old AF group.

Fig. 4 Representative immunoblots (Western blots) showing Ca2+ handling protein expression (LVDCCa1c, L-type calcium channel a1c; Ca2+-ATPase,

calcium adenosine triphosphatase; RYR2, ryanodine receptor type-2; IP3R1, inositol triphosphate receptor type-1; PLN, phospholamban) and b-actinin the LA myocardium between adult and aged groups in SR and AF. LA: left atira; SR: sinus rhythm; AF: atrial fibrillation; AS: Adult SR group; OS:Old SR group; AF: Adult AF group; OF: Old AF group.



phosphorylated and non-phosphorylated channels is shiftedtowards the non-phosphorylated state.

In conclusion, these age-associated electrophysiological andmolecular changes have suggested that ageing reduces the activity ofCa2+ handling proteins, abnormal Ca2+ handling might be due toimpaired phosphorylation-dependent regulation of these multi-stepCa2+ handling proteins, and this up-regulation tendency with ageing isprobably a physiological adaptation mechanism. AF is also associatedwith the elaborate adaptive and maladaptive reactions in the electro-physiology, functional ion-current and ion-channel gene and proteinexpression changes. The exact mechanism remains to be elucidated.

Study limitations

First, the plateau potential of AP was determined by IKur, Ito andICa-L, but our study did not included IKur and Ito. Second, APA isrelated with the INa, but we did not study the change in INa.Finally, our findings were limited to the studies of cardiomyocytesfrom LA and cannot necessarily be extended to cells of otherregions of the atria. Also, due to that the function of the intracel-lular Ca2+ handling protein was fully studied, it was not repeated

in the discussion. Ageing is also characterized by a progressivedeterioration in physiological functions and metabolic processes,which may alter the amount and distribution of ion channels. Fur-ther the extent to which the altered electrophysiological propertiesseen with ageing may be arrhythmogenic and increase the likeli-hood of AF is presently unknown.

Acknowledgements

This study was supported by the Program of National Natural Science Founda-

tion of China (no. 308660299), the Program of Natural Science Foundation of

the Xinjiang Uygur Autonomous Region (no. 200821143), the Program of Nat-

ural Science Foundation of the Xinjiang Uygur Autonomous Region (no.2011211A074), the Program of Doctoral Fund of Ministry of Education

(200807600004). The funders had no role in study design, data collection and

analysis, decision to publish or preparation of the manuscript.

Conflicts of interest

All authors confirm that there are no conflicts of interest.

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Age-related changes in cellular electrophysiology and calcium handling for atrial fibrillation